Gene expression patterns determine cell identity and are therefore essential for proper development and prevention of inappropriate cell proliferation. Expression patterns are determined by transcriptional activation and repression. Mechanisms of gene expression, the focus of this research project, can be local or regional and involve alterations in chromatin structure. Locus-specific repressors affect only a few nucleosomes, whereas regional silencing involves specialized chromatin structures that propagate great distances along chromosomes. Key issues in the field are how the formation of repressive chromatin is restricted in space and time and how these chromatin structures repress transcription. To address these issues, this proposal focuses on a non-spreading locus-specific repressor protein, Sum1p from S. cerevisiae and a mutant version of this protein, Sum1-1p, which propagates along a chromosome and achieves regional silencing. Sum1p and Sum1-1p differ only a single critical amino acid and therefore present a unique opportunity to explore the similarities and differences between local and regional repression. Using a combination of biochemical and genetic approaches to investigate the mechanisms by which these almost identical proteins have such different spreading properties, we will provide fundamental insights into the regulation of spreading in regional silencing as well as shared mechanism of repression. The 4 specific aims of this research project extend a considerable body of preliminary results. The aims are: (i) To understand how the SUM1-1 mutation changes the action of Sum1p by determing the function (s) of the conserved domain of Sum1p in which the amino acids resides, (ii) To better understand how wild-type and mutant Sum1p interact with chromatin by determining whether wild-type and/or mutant Sum1p bind to histones, (iii) To explore the role of an NAD+-dependent deacetylase in Sum1p-mediated repression, and (iv) To investigate how the spreading of Sum1-1p is regulated by studying the establishment of Sum1-1p-mediated silencing in vivo.